Efficient modification of the APRT gene by FLP/FRT site-specific targeting
The FLP/FRT site-specific recombination system was established and characterized at the APRT gene in CHO cells. Targeting frequencies with FLP-stimulation were about 1 to 5×10−5, which were 6–22-fold above gene targeting frequencies in the absence of FLP. Fifty two APRT+ cell lines were analyzed by Southern blotting: 56% were FLP-targeted integrants; 33% were APRT target convertants; 11% gave undefined patterns. In separate experiments we first enriched for integrants by screening for two additional markers carried on the targeting vector; 18 of 19 (95%) of the resulting cell lines were integrants. Intrachromosomal site-specific recombination was tested by reexposing integrants to FLP. Intrachromosomal popouts were stimulated over 200-fold, while homologous recombination in an adjacent interval was unchanged. The utility of this system was demonstrated by one-step FLP targeting to generate chromosomal substrates for homologous recombination, and by a two-step, FLP-and-run procedure to construct a chromosomal substrate for illegitimate recombination.
KeywordsGene Target Southern Blotting Homologous Recombination Separate Experiment Additional Marker
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